Fusion polypeptide having the C protein and E1 protein of hepatitis C virus

ABSTRACT

The invention concerns a pharmaceutical composition for treating or preventing C hepatitis (HCV), induced infections, which in a preferred embodiment, comprises a main active principle, (i) a fusion polypeptide, including the HCV capsid polypeptide (C191) and polypeptide coat (E1) and in which at least one cleavage site 173/174 and 191/192 has been made inoperative by mutation; (ii) an equimolar mixture of the C191 polypeptide of which the cleavage site 173/174 has been made inoperative and of the E1 polypeptide (mixture equivalent to the fusion polypeptide); or (iii) a DNA molecule coding for this fusion polypeptide. Products (i) to (iii) are characterized in that the C191 element is incapable of regulating the functioning of the genes, in particular of causing them to interact. Such a composition can also include any form equivalent to the products described above.

RELATED APPLICATION(S)

This application is a Continuation-In-Part of and claims priority to PCT/FR98/00448, filed on Mar. 6, 1998, which claims priority to French application 97/02,887, filed on Mar. 6, 1997, the entire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The invention relates to a pharmaceutical composition intended for the treatment or prophylaxis of infections induced by the hepatitis C virus (HCV).

The hepatitis C virus (HCV) is the agent responsible for the majority of hepatitis infections of the non-A non-B type. The seroprevalence of HCV infections varies between 0.3 and 1.5% in the world population, possibly reaching 18% in some developing countries. Hundreds of millions of people are thus thought to be infected worldwide. Nine types and thirty subtypes of HCV have been described. The subtypes may be associated with a defined geographical distribution, type 1b being the most widespread worldwide. The progression to the chronic form occurs in 50% of cases, about 5 years after the primary infection. Persistent Chronic Hepatitis which is asymptomatic, but which exhibits a high circulating virus titer, is first observed, then Active Chronic Hepatitis becomes established. Twenty percent of chronic hepatites progress to sclerosis of the liver within about ten years. Hepatocarcinoma may develop in the cirrhotic liver.

The hepatitis C virus (HCV) is a positive single-stranded RNA virus. On the basis of structural resemblance, HCV has been linked to the flavivirus and pestivirus families.

SUMMARY OF THE INVENTION

The present invention relates to fusion polypeptide that comprises a first region having the C polypeptide of the hepatitis C virus (HCV) or a portion thereof that comprises a polypeptide region responsible for gene regulatory activity; and a second region having the envelope polypeptide (E1) of the virus or a portion thereof that comprises a site for cytoplasmic anchorage of the E1 polypeptide, wherein the first region is fused by a peptide bond to the second region, and the fusion polypeptide is not cleaved by a mammalian protease.

The present invention also relates to a fusion polypeptide comprising a C polypeptide of HCV or a portion thereof and an E1 envelope polypeptide of HCV or a portion thereof. The C polypeptide comprises a first C polypeptide region responsible for gene regulatory activity and a second C polypeptide region responsible for the interaction with the E1 envelope polypeptide, wherein the site of interaction with the E1 polypeptide is between about 151 and about 173, or between about 173 and about 191. The E1 envelope polypeptide comprises a first E1 polypeptide region responsible for E1 cytoplasmic anchorage and a second E1 polypeptide region responsible for the interaction of the second C polypeptide region. The site for interaction with the C polypeptide is between about amino acid 330 and about amino acid 380. The C polypeptide is fused by a peptide bond to the E1 envelope polypeptide, and the C polypeptide comprises Cysteine₁₇₂Serine₁₇₃-Phenylalanine₁₇₄-Serine₁₇₅ with at least one mutation between amino acid Nos 172 and 175.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the HCV genome which consists of RNA with its untranslated 5′ and 3′ regions indicated by lines, and the open reading frame of the precursor polyprotein indicated in the form of a rectangle.

FIG. 2 represents the inserts derived from the HCV genome which are tested in plasmids pRC. The sequences derived from the HCV genome are represented by a rectangle and the mutated residues are indicated by dots.

FIG. 3 is a diagram representing the luciferase activity measured for each of the constructs of which some are mutated at the level of one or more cleavage sites. The identity of the insert tested appears on the x-axis while the quantity of luciferase produced relative to the total quantity of protein produced appears on the y-axis.

FIGS. 4A-4L are an illustration of the nucleic acid sequence (SEQ ID NO.:1) and amino acid sequence (SEQ ID NO.:2) of HCV.

DETAILED DESCRIPTION OF THE INVENTION

During an infectious event, the HCV genome is first translated into a precursor polyprotein of about 3000 amino acids. This polyprotein then undergoes post-translational cleavages to give various precursors and mature viral proteins. The structural proteins of HCV are located in the N-terminal region of the polyprotein. As shown in FIG. 1, they are more particularly the capsid or core protein (C), and the envelope proteins E1 and E2, which are present in the following order: NH2-C-E1-E2. This portion is cleaved by the host cell proteases.

The numbering of the amino acids of the polyprotein as well as of its derivatives, which is adopted here after, is that commonly used and in particular presented by Choo et al., PNAS [vol. 88: p.2451 (1991)]. Thus, the C protein corresponds to the amino acids at positions 1 to 191 of the polyprotein and the E1 protein to the amino acids at positions 192 to 380. In the remainder of the text, it is appropriate to number the amino acids of the sequence of the E1 protein, from position 192 to position 380, 381, 382 or 383. In the remainder of the text, for the sake of simplicity, reference is made solely to the C-terminal position 380.

The C protein derived from the direct cleavage of the polyprotein contains 191 amino acids. This C protein, also called C191, may itself be truncated toward its C-terminal end by enzymatic cleavage to give a protein of 173 amino acids, called C173. In the remainder of the text, the term “C protein or polypeptide” will preferably designate the C191 form.

The C protein is a good vaccine candidate since it is of course a structural protein of the virus and since the region encoding this protein is relatively well conserved by the various HCV strains. It is known that a region of the C protein capable of generating a high antibody response corresponds to the first 120 amino acids; the first 48 amino acids constituting the major antigenic domain. However, a major obstacle to its use as vaccine lies in the fact that this protein is capable of transactivating genes belonging to the host cell, in particular genes such as oncogenes, which may have, inter alia, the consequence of inducing a carcinogenesis event.

Indeed, it has in particular been shown that the C173 form was capable of translocation in the nucleus of the host cell and of transactivation. The region of the C protein responsible for the translocation in the nucleus and for the regulatory activity appears to be located in the N-terminal portion (first 123 amino acids).

Thus, the region of the C protein which is of interest from a vaccine point of view is, on the other hand, responsible for a toxic effect toward the host cell.

To overcome this difficulty, a solution commonly envisaged in the scientific community would be to use a C191 protein whose cleavage site at position 173/174 would have been made inoperative by mutation. As will be seen below, such a protein nevertheless proves capable of regulatory activity, even if it is to a lesser degree.

Surprisingly, it has now been demonstrated that it was possible to abolish the regulatory activity of the C protein by modifying it and by combining it, under certain conditions, with the E1 protein. The present invention provides means for abolishing the regulatory activity by preventing the migration of the C protein into the nucleus. This migration no longer takes place in the presence of the E1 protein which possesses, inter alia, the property of becoming anchored in the cytoplasm, at the level of the endoplasmic reticulum, and which, unexpectedly, has the capacity to retain the C protein therein when certain conditions are met. The migration may be abolished by producing, for example, a fusion of the two proteins, cleavable or otherwise; in the case where it is cleavable, the products generated should be capable of interacting with each other so that there is no leakage of one of them into the nucleus; the complex formed by the product of cleavage being capable of becoming anchored in the cytoplasm. The equivalent of a cleavable peptide fusion is a mixture, in equimolar quantity, of the components constituting the fusion.

Accordingly, the subject of the invention is a pharmaceutical composition comprising:

(i) A polypeptide which contains:

(a) a first region corresponding to all or part of the C polypeptide of the hepatitis C virus; and

(b) a second region corresponding to all or part of the E1 polypeptide of said virus and,

proves, as such or via its products of cleavage, incapable of regulatory activity toward one or more genes;

(ii) A mixture (preferably) in substantially equimolar quantity,

(a) of a first polypeptide containing a region which corresponds to all or part of the C polypeptide of HCV and

(b) of a second polypeptide containing a region corresponding to all or part of the E1 polypeptide of HCV; and

which proves incapable of regulatory activity toward one or more genes; or

(iii) A DNA molecule comprising a sequence encoding the polypeptide as described in (i) of the present claim, placed under the control of elements necessary for its expression in a mammalian cell; and

a pharmaceutically acceptable carrier or diluent.

According to another aspect of the invention, the subject of the invention is also a method for the treatment or prevention of an infection induced by HCV according to which a pharmaceutical composition according to the invention is administered to a mammal, preferably a human, requiring such a treatment.

“Polypeptide” is understood to mean any chain of amino acids covalently linked to each other, regardless of the length of the chain and regardless of the post-translational modifications which may take place such as, for example, a lipidation. It is also possible to use the term protein interchangeably.

“C polypeptide of HCV” is understood to mean in particular a C polypeptide which possesses the amino acid sequence as disclosed by Choo et al. as well as any other C polypeptide obtained from any other strain and whose sequence could differ from that of Choo et al. For example, it may represent the C polypeptides described by Takeuchi K et al. [Nucleic Acids Research 18:4626 (1990)], Houghton M et al. [Hepatology 14:381 (1991)], Delisse et al. [J. Hepatology, 13, suppl. 4 (1991)], Bukh J et al. [PNAS 91:8239 (1994)] and Hiroaki O et al. [Intervirology 37:68 (1994)].

“E1 polypeptide of HCV” is understood to mean in particular an E1 polypeptide which possesses the amino acid sequence as disclosed by Choo et al. as well as any other E1 polypeptide obtained from any other strain and whose sequence could differ from that of Choo et al. For example, it may represent the E1 polypeptides described in Hiroaki O et al. [Intervirology 37:68 (1994)], Grakoui et al. [J. Virol. 67:1385 (1993)] and Spaete et al. [Virology 188:819 (1992)], Matsumia et al. [J. Virol. 66:1425] or in Kohara et al. [J. Gen. Virol. 73:2313 (1992)].

Thus, the amino acid sequence of the C polypeptide and that of the E1 polypeptide of HCV may vary according to the viral strain, reflecting the phenomenon of allelic variance. For example, a virus is usually represented by a set of strains which differ from each other in minor allelic characteristics. A polypeptide which fulfills the same biological function in different strains may have an amino acid sequence which is not the same for all the strains. Such an allelic variation is also found at the level of the DNA.

At the level of the amino acid sequence, the allelic differences may consist of one or more amino acid substitutions, deletions or additions which do not alter the biological function.

As regards the polypeptide included in the pharmaceutical composition according to the invention, two cases must be envisaged: either the polypeptide is incapable of being cleaved by a protease in a mammalian cell, or it is susceptible to such a cleavage.

When the polypeptide is incapable of being cleaved by a protease in a mammalian cell, it advantageously contains:

(a) a first region corresponding at least to the portion of the C polypeptide of the HCV virus responsible for the regulatory activity of said C polypeptide toward one or more genes; and

(b) a second region corresponding at least to a portion of the E1 polypeptide of said virus responsible for the cytoplasmic anchorage of the E1 polypeptide.

When the polypeptide is capable of being cleaved by a protease in a mammalian cell, it advantageously contains:

(a) a first region corresponding at least to a portion of the C polypeptide of HCV responsible for the regulatory activity of said C polypeptide toward one or more genes and to the portion of said C polypeptide responsible for the interaction of said C polypeptide with the E1 polypeptide of said virus; and

(b) a second region corresponding at least to a portion of the E1 polypeptide of said virus responsible for the interaction of the E1 polypeptide with the C polypeptide of said virus and to a portion of the E1 polypeptide of said virus responsible for the cytoplasmic anchorage of the E1 polypeptide.

“Portion of the C polypeptide of HCV responsible for the regulatory activity of said C polypeptide toward one or more genes” is understood to mean in particular any portion of the C polypeptide of HCV capable of activating, transactivating or suppressing the transcription or the expression of any gene, according to any mechanism. This gene may be a eukaryotic gene, a viral gene, an oncogene or a protooncogene.

A portion of the C polypeptide of HCV responsible for the regulatory activity of said polypeptide may in particular correspond to the amino acids at positions 38 to 43, 58 to 64, 66 to 71, 6 to 23, 39 to 74, 99 to 102, 101 to 121, 101 to 122, 58 to 121, 1 to 120, 1 to 121, 1 to 122, 1 to 123 or 1 to 173. Preferably, a portion of the C polypeptide of HCV responsible for the regulatory activity may be a portion of the C polypeptide ranging from the amino acid at position 1 to the amino acid in one of positions 48 to 191. For this purpose, one of positions 48 to 191 may be for example position 119, 120, 121, 123 and 173.

A portion of the C polypeptide of HCV responsible for the interaction of said C polypeptide with the E1 protein of HCV may in particular correspond to the amino acids at positions 151 to 173 or at positions 173 to 191 of the C polypeptide of HCV.

A portion of the E1 polypeptide of HCV responsible for the cytoplasmic anchorage of the E1 polypeptide may be a hydrophobic domain of the E1 polypeptide. Such hydrophobic domains are for example located at positions 262 to 291, 370 to 380 and 330 to 380 of the E1 polypeptide.

A portion of the E1 polypeptide of HCV responsible for the interaction of the C polypeptide with the E1 protein may be in particular the C-terminal domain of the E1 polypeptide, preferably the domain at positions 330 to 380 or at positions 370 to 380.

In a polypeptide useful for the purposes of the present invention, the first region may be located on the N- or C-terminal side of the polypeptide, advantageously on the N-terminal side; likewise, the second region may be located on the C- or N-terminal side, advantageously on the C-terminal side. According to a preferred mode, the C-terminal end of the first region may be fused by peptide bonding to the N-terminal end of the second region.

When the polypeptide contained in the pharmaceutical composition according to the invention comprises the region corresponding at least to the amino acids at positions 172 to 175 of the C polypeptide of HCV, this polypeptide advantageously contains a mutation making the cleavage site at position 173/174 inoperative. According to a preferred mode, such a mutation is a point mutation, carried out in one of positions 172 to 175. It may be obtained, for example, by deletion, addition or substitution of one or more amino acids, in particular by deletion, addition or substitution of one or more amino acid at positions 172 to 175. Preferably the mutation will be produced by substitution of one or two amino acids; a double mutation by substitution being most particularly preferred. According to a particular example, the residue naturally existing at position 173 (serine) may be in particular substituted by the methionine residue and the residue naturally existing at position 173 (phenylalanine) may be substituted in particular by the leucine residue. In general, it is within the capability of persons skilled in the art to produce one or more mutations capable of making inoperative the cleavage site at position 173/174 of the C polypeptide of HCV.

When the polypeptide contained in the pharmaceutical composition according to the invention comprises the region corresponding at least to the amino acids at positions 190 to 193 of the HCV polyprotein, this polypeptide advantageously contains a mutation making inoperative the cleavage site at position 191/192 of the HCV polyprotein. According to a preferred mode, such a mutation is a point mutation produced in one of positions 190 to 193. It may be obtained, for example, by deletion, addition or substitution of one or more amino acids, in particular by deletion, addition or substitution of one or more amino acids at positions 190 to 193. Preferably, the mutation will be produced by substitution of one or two amino acids, a double mutation by substitution being most particularly preferred. According to a specific example, the residue naturally existing at position 191 (alanine) may in particular be substituted by the valine residue and the residue naturally existing at position 192 (tyrosine) may in particular be substituted by the asparagine residue. In general, it is within the capability of persons skilled in the art to produce one or more mutations capable of making inoperative the cleavage site at position 191/192.

When the polypeptide useful for the purposes of the present invention comprises both the region corresponding at least to amino acids 190 to 193 of the HCV polyprotein and the region corresponding at least to amino acids 172 to 175 of the C polypeptide of HCV, only one of the two cleavage sites 191/192 and 173/174 can be made inoperative, preferably both will be made inoperative. When only the site 173/174 is made inoperative, the polypeptide is capable of being cleaved and in this particular case, it is necessary that this polypeptide possesses a first region which corresponds, inter alia, to the portion of the C polypeptide responsible for the interaction of said polypeptide with the E1 polypeptide and a second region which corresponds, inter alia, to the portion of the E1 polypeptide responsible for the interaction of said polypeptide with the C polypeptide.

When a polypeptide useful for the purposes of the present invention is incapable of being cleaved by a protease, it may contain a cleavage site on the condition, however, that this cleavage site is not functional. For example, in the particular case of a polypeptide consisting of the C191 polypeptide fused with the E1 polypeptide and containing a mutation making inoperative the cleavage site at position 191/192, the cleavage site at position 173/174 may not be mutated; however, it will not be, or will be only slightly, functional, insofar as the cleavage at position 191/192 is no longer possible. Indeed, it is known that the cleavage at position 191/192 must be carried out for the cleavage at position 173/174 to take place.

According to a specific mode, a polypeptide useful for the purposes of the present invention is incapable of being cleaved by a protease and contains:

(a) a first region which substantially corresponds to the domain of the C polypeptide ranging from the amino acid at position 1 to the amino acid in one of positions 120 to 173, and

(b) a second region which substantially corresponds to a domain of the E1 polypeptide containing at least one hydrophobic region, for example to the domain of the E1 polypeptide ranging from the amino acid at position 192 to the amino acid at position 380, or from the amino acid at position 330 to the amino acid at position 380, or from the amino acid at position 260 to the amino acid at position 290, or from the amino acid at position 260 to the amino acid at position 380.

According to another particular mode, a polypeptide useful for the purposes of the present invention is incapable of being cleaved by a protease and contains:

(a) a first region which substantially corresponds to the domain of the C polypeptide ranging from the amino acid at position 1 to the amino acid in one of positions 120 to 191, and

(b) a second region which substantially corresponds to a domain of the E1 polypeptide containing at least one hydrophobic region, for example to the domain of the E1 polypeptide ranging from the amino acid at position 192 to the amino acid at position 380, or from the amino acid at position 330 to the amino acid at position 380, or from the amino acid at position 260 to the amino acid at position 290, or from the amino acid at position 260 to the amino acid at position 380;

on the condition that said polypeptide does not contain a cleavage site 191/192 or alternatively when the cleavage site is reconstituted, then a mutation is introduced in order to make it inoperative.

Advantageously, the first region of the polypeptide useful for the purposes of the present invention corresponds to the amino acids at positions 1 to 191 of the C polypeptide of HCV and/or the second region of this polypeptide corresponds at least to the amino acids at positions 192 to 380 of the E1 polypeptide or HCV. In a particularly preferred manner, the first and second regions are as defined above in this same paragraph, the amino acid at position 191 being fused by peptide bonding to the amino acid at position 192. According to a particular mode, the polypeptide consists of the first and second regions as defined above in this same paragraph.

When the polypeptide useful for the purposes of the present invention is as described in the preceding paragraph, it imperatively contains a mutation making inoperative the cleavage site at position 191/192 or at position 173/174. Preferably, the two cleavage sites are made inoperative.

A mixture useful for the purposes of the present invention advantageously comprises:

(a) a first polypeptide containing a region which corresponds at least to the portion of the C polypeptide of the HCV virus responsible for the regulatory activity of said C polypeptide toward one or more genes and to the portion of said C polypeptide responsible for the interaction of said C polypeptide with the E1 polypeptide of said virus, and

(b) a second polypeptide containing a region corresponding to a portion of the E1 polypeptide of said virus responsible for the interaction of the E1 polypeptide with the C polypeptide of said virus and to a portion of the E1 polypeptide of said virus responsible for the cytoplasmic anchorage of the E1 polypeptide.

The portions of the C and E1 polypeptides responsible for the properties listed in points (a) and (b) of the preceding paragraph may be as described above for the fusion polypeptide.

Preferably, the first polypeptide of the mixture contains and in a most particularly preferred manner consists of a region corresponding to the amino acids at positions 1 to 191 of the C polypeptide (C191) of HCV. In the latter case, the cleavage site at position 173/174 must be made inoperative by mutation. This mutation may be produced as described above for the fusion polypeptide.

Preferably, the second polypeptide of the mixture contains and in a most particularly preferred manner consists of a region corresponding to the amino acids at positions 192 to 380 of the E1 polypeptide of HCV.

For the purposes of the present invention, a DNA molecule may be a simple linear DNA fragment, or alternatively a plasmid or alternatively a viral vector such as a pox vector.

A polypeptide, a mixture or a molecule of DNA as described in the present application are of a most special interest when they are used for the manufacture of a medicament intended for the treatment or prevention of infections induced by HCV. They are in particular useful in the immunotherapy of infections induced by HCV, most particularly a DNA molecule.

Finally, the invention relates to a method for inducing an immune response toward HCV in a mammal, according to which an immunologically effective quantity of a composition according to the invention is administered to said mammal in order to develop an immune response. The invention also relates to a method for the prevention or treatment of an infection induced by HCV, according to which a prophylactically or therapeutically effective quantity of a composition according to the invention is administered to an individual.

The methods and the pharmaceutical compositions according to the invention can treat or prevent HCV infections and consequently hepatic diseases associated with such infections. They are in particular persistent chronic hepatitis, active chronic hepatitis, cirrhosis of the liver and hepatocarcinomas.

A composition according to the invention may be administered by any conventional route used in the field of vaccines, in particular by the parenteral (e.g. subcutaneous, intradermal, intramuscular, intravenous or intraperitoneal) route. The choice of the route of administration depends on a number of parameters such as the nature of the active principle, polypeptide or DNA molecule, the adjuvant combined with the polypeptide or with the DNA molecule.

A composition according to the invention may comprise, in addition to a polypeptide or a mixture of polypeptides useful for the purposes of the present invention, at least one other HCV antigen such as the E2 protein or alternatively such as a nonstructural protein NS1, NS2, NS3, NS4 or NS5, or a subunit, fragment, homolog, mutant or derivative of these antigens.

A polypeptide, a mixture or a molecule of DNA useful for the purposes of the present invention may be formulated in or with liposomes, preferably neutral or anionic liposomes, microspheres ISCOMs or virus-like particles (VLPs), in order to promote the screening of the protein or of the polypeptide or to increase the immune response. Persons skilled in the art have these compounds available without difficulty; for example see Liposomes: A Practical Approach. RRC New ED, IRL press (1990).

Adjuvants other than liposomes may also be used. A large number are known to persons skilled in the art. Such adjuvants are identified by references below:

For parenteral administration, there may be mentioned in particular aluminum compounds such as aluminum hydroxide, aluminum phosphate and aluminum hydroxyphosphate. The antigen may be absorbed or precipitated on an aluminum compound according to standard methods. Other adjuvants useful for parenteral administration include in particular polyphosphazene (WO 95/2415), DC-chol (3-beta-[N-(N′, N′-dimethylaminomethane)carbamoyl]cholesterol) (U.S. Pat. No. 5,283,185 and WO 96/14831), QS-21 (WO 88/9336) and RIBI from ImmunoChem (Hamilton, Mont.).

The administration may take place in a single dose or in a dose repeated once or several times after a certain period. The appropriate dosage varies according to various parameters, for example the individual treated (adult or child), the vaccinal antigen itself, the mode and frequency of administration, the presence or absence of adjuvant and, if present, the type of adjuvant and the desired effect (e.g. protection or treatment), as will be determined by persons skilled in the art.

A composition according to the invention may be manufactured conventionally. In particular, a polypeptide, a mixture or a molecule of DNA contained in the composition according to the invention is combined with a pharmaceutically acceptable diluent or carrier, e.g. water or a saline solution such as phosphate-buffered saline (PBS). In general, the diluent or the carrier is selected on the basis of the mode and route of administration and of standard pharmaceutical practices. Pharmaceutically acceptable diluents and carriers as well as all that is necessary for their use in pharmaceutical formulations are described in Remington's Pharmaceutical Sciences, a standard reference text in this field and in USP/NP.

EXEMPLIFICATION EXAMPLE 1 Construction of Recombinant Plasmids and Site-Directed Mutagenesis

The constructs called pRC/E1, pRC/CE1M1, pRC/CE1M2 and pRC/CE1M1M2 (also call pRC/CE1DM), pRC/C191M1 and pRC/C173, which are used below in Example 2, have been described in: Liu Q et al., J. Virol. 71: 657 (1997). The inserts used are represented in FIG. 2. All the constructs are produced in the vector pRC which is obtained from InVitrogen (ref: V780-20). The vector pRC carries the ampicillin gene and allows the expression of inserts under the control of a CMV promoter. Mutations called M1 and M2 are present in the constructs pRC/C191M1, pRC/CE1M1, pRC/CE1M2 and pRC/CE1M1M2. They were generated by site-directed mutagenesis performed by PCR. The mutation called M1 corresponds to the replacement of the amino acids Serine¹⁷³ and Phe¹⁷⁴ of the C protein with the amino acids methionine and leucine, respectively. The mutation called M2 corresponds to the replacement of the amino acids alanine¹⁹¹ and tyrosine¹⁹² of the CE1 protein with the amino acids valine and asparagine, respectively.

The plasmids expressing the reporter genes for luciferase and for β-galactosidase were constructed by modifying the vector pUC 18 (Appligene; ref: 161131). The expression of the genes is under the control of the immediate-early promoter 1(ie1) of the human CMV. Sequences derived from the 3′ region of the bovine gene for the growth hormone were moreover added in the 3′ of the genes in order to stabilize the mRNAs. These plasmids carry more than one ampicillin gene.

EXAMPLE 2 Transfection of Cells with the Plasmids

CHO-K1 cells (ATCC CCL 61) were stored in α-MEM medium [Nature 230:310 (1971)], supplemented with 10% Foetal Calf Serum (FCS) (Hyclone, ref:A1115-L) and 20% Dimethyl Sulfoxide (DMSO) in liquid nitrogen. These cells are cultured under humid atmosphere at 37° C. with 5% CO₂ and 95% air. To carry out subcultures, the medium removed and the cellular lawn is rinsed with 5 ml of phosphate buffer (PBS). The supernatant is then removed before addition of 1.5 ml of trypsine per 75 cm² flask (trypsine at 0.025%). After incubating for 10 min in an incubator at 37° C., the reaction is stopped by addition of 10 ml of α-MEM medium containing 10% FCS. The cells are counted on a Malassez cell after a one-half dilution in 0.02% Trypan blue. 5×10⁵ cells are then inoculated into dishes 6 cm in diameter with complete medium.

The CHO cells are then cotransfected with one of the recombinant plasmids (pHCV) described above and a reporter plasmid (pCMV) which contains either the β-galactosidase gene under the control of the CMV promoter (pCMV β-gal) or the luciferase gene under the control of the CMV promoter (pCMV Luc).

For that, 5 μg of DNA (4.5 μg of plasmid pHCV/0.5 μg of plasmid pCMV) are diluted in 500 μl of OPTI-MEM medium (Gibco), and mixed with 14 μl of lipofectamine diluted in 500 μl of the same medium. The two solutions are mixed and incubated for 20 min at room temperature in order to allow the formation of the DNA-liposome complexes.

The DNA liposome mixture diluted with 2 ml of OPTI-MEM is then added to the cells after removing the culture medium and rinsing in PBS. After incubating for 5 hours, the medium is again changed and 48 h after the transfection, it is then possible to test for the transient expression of the recombinant genes.

EXAMPLE 3 Demonstration of the Regulatory Activity of the Constructs on Reporter Genes

The transfected cells are lysed with the aid of the reagent “Luciferase Cell Culture Lysis Reagent” (Promega, Luciferase Assay System). 100 μl of substrate are added to 100 μl of cell supernatant, directly by the bioluminometer injector (Lumat LB/9501/16 from Berthold) which measures the quantity of light emitted (Relative Light Units) for 10 seconds. The quantity of light emitted is then converted to nanograms of proteins per ml of cell lysate, by comparing with a standard curve established with the aid of purified luciferase.

The results, which are presented in FIG. 2, show that a point mutation at the amino acid 191 in the construct CE1M2 abolishes the transactivating effect. A point mutation at the amino acid 173 (in the construct CE1M1) abolishes the transactivating effect only in the case where C is fused with E1. A double mutation at the amino acids 173 and 191 abolishes the transactivating effect.

The teachings of all the articles, patents, and patent applications cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

2 1 9379 DNA Virus CDS (320)...(9352) 1 cactccacca tgaatcactc ccctgtgagg aactactgtc ttcacgcaga aagcgtctag 60 ccatggcgtt agtatgagtg tcgtgcagcc tccaggaccc cccctcccgg gagagccata 120 gtggtctgcg gaaccggtga gtacaccgga attgccagga cgaccgggtc ctttcttgga 180 tcaacccgct caatgcctgg agatttgggc gtgcccccgc aagactgcta gccgagtagt 240 gttgggtcgc gaaaggcctt gtggtactgc ctgatagggt gcttgcgagt gccccgggag 300 gtctcgtaga ccgtgcacc atg agc acg aat cct aaa cct caa aaa aaa aac 352 Met Ser Thr Asn Pro Lys Pro Gln Lys Lys Asn 1 5 10 aaa cgt aac acc aac cgt cgc cca cag gac gtc aag ttc ccg ggt ggc 400 Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly 15 20 25 ggt cag atc gtt ggt gga gtt tac ttg ttg ccg cgc agg ggc cct aga 448 Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg 30 35 40 ttg ggt gtg cgc gcg acg aga aag act tcc gag cgg tcg caa cct cga 496 Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg 45 50 55 ggt aga cgt cag cct atc ccc aag gct cgt cgg ccc gag ggc agg acc 544 Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr 60 65 70 75 tgg gct cag ccc ggg tac cct tgg ccc ctc tat ggc aat gag ggc tgc 592 Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys 80 85 90 ggg tgg gcg gga tgg ctc ctg tct ccc cgt ggc tct cgg cct agc tgg 640 Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp 95 100 105 ggc ccc aca gac ccc cgg cgt agg tcg cgc aat ttg ggt aag gtc atc 688 Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile 110 115 120 gat acc ctt acg tgc ggc ttc gcc gac ctc atg ggg tac ata ccg ctc 736 Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu 125 130 135 gtc ggc gcc cct ctt gga ggc gct gcc agg gcc ctg gcg cat ggc gtc 784 Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val 140 145 150 155 cgg gtt ctg gaa gac ggc gtg aac tat gca aca ggg aac ctt cct ggt 832 Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly 160 165 170 tgc tct ttc tct atc ttc ctt ctg gcc ctg ctc tct tgc ttg act gtg 880 Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val 175 180 185 ccc gct tcg gcc tac caa gtg cgc aac tcc acg ggg ctt tac cac gtc 928 Pro Ala Ser Ala Tyr Gln Val Arg Asn Ser Thr Gly Leu Tyr His Val 190 195 200 acc aat gat tgc cct aac tcg agt att gtg tac gag gcg gcc gat gcc 976 Thr Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Ala 205 210 215 atc ctg cac act ccg ggg tgc gtc cct tgc gtt cgt gag ggc aac gcc 1024 Ile Leu His Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ala 220 225 230 235 tcg agg tgt tgg gtg gcg atg acc cct acg gtg gcc acc agg gat ggc 1072 Ser Arg Cys Trp Val Ala Met Thr Pro Thr Val Ala Thr Arg Asp Gly 240 245 250 aaa ctc ccc gcg acg cag ctt cga cgt cac atc gat ctg ctt gtc ggg 1120 Lys Leu Pro Ala Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly 255 260 265 agc gcc acc ctc tgt tcg gcc ctc tac gtg ggg gac cta tgc ggg tct 1168 Ser Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser 270 275 280 gtc ttt ctt gtc ggc caa ctg ttc acc ttc tct ccc agg cgc cac tgg 1216 Val Phe Leu Val Gly Gln Leu Phe Thr Phe Ser Pro Arg Arg His Trp 285 290 295 acg acg caa ggt tgc aat tgc tct atc tat ccc ggc cat ata acg ggt 1264 Thr Thr Gln Gly Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly 300 305 310 315 cac cgc atg gca tgg gat atg atg atg aac tgg tcc cct acg acg gcg 1312 His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala 320 325 330 ttg gta atg gct cag ctg ctc cgg atc cca caa gcc atc ttg gac atg 1360 Leu Val Met Ala Gln Leu Leu Arg Ile Pro Gln Ala Ile Leu Asp Met 335 340 345 atc gct ggt gct cac tgg gga gtc ctg gcg ggc ata gcg tat ttc tcc 1408 Ile Ala Gly Ala His Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser 350 355 360 atg gtg ggg aac tgg gcg aag gtc ctg gta gtg ctg ctg cta ttt gcc 1456 Met Val Gly Asn Trp Ala Lys Val Leu Val Val Leu Leu Leu Phe Ala 365 370 375 ggc gtc gac gcg gaa acc cac gtc acc ggg gga agt gcc ggc cac act 1504 Gly Val Asp Ala Glu Thr His Val Thr Gly Gly Ser Ala Gly His Thr 380 385 390 395 gtg tct gga ttt gtt agc ctc ctc gca cca ggc gcc aag cag aac gtc 1552 Val Ser Gly Phe Val Ser Leu Leu Ala Pro Gly Ala Lys Gln Asn Val 400 405 410 cag ctg atc aac acc aac ggc agt tgg cac ctc aat agc acg gcc ctg 1600 Gln Leu Ile Asn Thr Asn Gly Ser Trp His Leu Asn Ser Thr Ala Leu 415 420 425 aac tgc aat gat agc ctc aac acc ggc tgg ttg gca ggg ctt ttc tat 1648 Asn Cys Asn Asp Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr 430 435 440 cac cac aag ttc aac tct tca ggc tgt cct gag agg cta gcc agc tgc 1696 His His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys 445 450 455 cga ccc ctt acc gat ttt gac cag ggc tgg ggc cct atc agt tat gcc 1744 Arg Pro Leu Thr Asp Phe Asp Gln Gly Trp Gly Pro Ile Ser Tyr Ala 460 465 470 475 aac gga agc ggc ccc gac cag cgc ccc tac tgc tgg cac tac ccc cca 1792 Asn Gly Ser Gly Pro Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pro 480 485 490 aaa cct tgc ggt att gtg ccc gcg aag agt gtg tgt ggt ccg gta tat 1840 Lys Pro Cys Gly Ile Val Pro Ala Lys Ser Val Cys Gly Pro Val Tyr 495 500 505 tgc ttc act ccc agc ccc gtg gtg gtg gga acg acc gac agg tcg ggc 1888 Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly 510 515 520 gcg ccc acc tac agc tgg ggt gaa aat gat acg gac gtc ttc gtc ctt 1936 Ala Pro Thr Tyr Ser Trp Gly Glu Asn Asp Thr Asp Val Phe Val Leu 525 530 535 aac aat acc agg cca ccg ctg ggc aat tgg ttc ggt tgt acc tgg atg 1984 Asn Asn Thr Arg Pro Pro Leu Gly Asn Trp Phe Gly Cys Thr Trp Met 540 545 550 555 aac tca act gga ttc acc aaa gtg tgc gga gcg cct cct tgt gtc atc 2032 Asn Ser Thr Gly Phe Thr Lys Val Cys Gly Ala Pro Pro Cys Val Ile 560 565 570 gga ggg gcg ggc aac aac acc ctg cac tgc ccc act gat tgc ttc cgc 2080 Gly Gly Ala Gly Asn Asn Thr Leu His Cys Pro Thr Asp Cys Phe Arg 575 580 585 aag cat ccg gac gcc aca tac tct cgg tgc ggc tcc ggt ccc tgg atc 2128 Lys His Pro Asp Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile 590 595 600 aca ccc agg tgc ctg gtc gac tac ccg tat agg ctt tgg cat tat cct 2176 Thr Pro Arg Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro 605 610 615 tgt acc atc aac tac acc ata ttt aaa atc agg atg tac gtg gga ggg 2224 Cys Thr Ile Asn Tyr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly 620 625 630 635 gtc gaa cac agg ctg gaa gct gcc tgc aac tgg acg cgg ggc gaa cgt 2272 Val Glu His Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg 640 645 650 tgc gat ctg gaa gac agg gac agg tcc gag ctc agc ccg tta ctg ctg 2320 Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu 655 660 665 acc act aca cag tgg cag gtc ctc ccg tgt tcc ttc aca acc cta cca 2368 Thr Thr Thr Gln Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro 670 675 680 gcc ttg tcc acc ggc ctc atc cac ctc cac cag aac att gtg gac gtg 2416 Ala Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val 685 690 695 cag tac ttg tac ggg gtg ggg tca agc atc gcg tcc tgg gcc att aag 2464 Gln Tyr Leu Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys 700 705 710 715 tgg gag tac gtc gtt ctc ctg ttc ctt ctg ctt gca gac gcg cgc gtc 2512 Trp Glu Tyr Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val 720 725 730 tgc tcc tgc ttg tgg atg atg cta ctc ata tcc caa gcg gag gcg gct 2560 Cys Ser Cys Leu Trp Met Met Leu Leu Ile Ser Gln Ala Glu Ala Ala 735 740 745 ttg gag aac ctc gta ata ctt aat gca gca tcc ctg gcc ggg acg cac 2608 Leu Glu Asn Leu Val Ile Leu Asn Ala Ala Ser Leu Ala Gly Thr His 750 755 760 ggt ctt gta tcc ttc ctc gtg ttc ttc tgc ttt gca tgg tat ttg aag 2656 Gly Leu Val Ser Phe Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Lys 765 770 775 ggt aag tgg gtg ccc gga gcg gtc tac acc ttc tac ggg atg tgg cct 2704 Gly Lys Trp Val Pro Gly Ala Val Tyr Thr Phe Tyr Gly Met Trp Pro 780 785 790 795 ctc ctc ctg ctc ctg ttg gcg ttg ccc cag cgg gcg tac gcg ctg gac 2752 Leu Leu Leu Leu Leu Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp 800 805 810 acg gag gtg gcc gcg tcg tgt ggc ggt gtt gtt ctc gtc ggg ttg atg 2800 Thr Glu Val Ala Ala Ser Cys Gly Gly Val Val Leu Val Gly Leu Met 815 820 825 gcg ctg act ctg tca cca tat tac aag cgc tat atc agc tgg tgc ttg 2848 Ala Leu Thr Leu Ser Pro Tyr Tyr Lys Arg Tyr Ile Ser Trp Cys Leu 830 835 840 tgg tgg ctt cag tat ttt ctg acc aga gtg gaa gcg caa ctg cac gtg 2896 Trp Trp Leu Gln Tyr Phe Leu Thr Arg Val Glu Ala Gln Leu His Val 845 850 855 tgg att ccc ccc ctc aac gtc cga ggg ggg cgc gac gcc gtc atc tta 2944 Trp Ile Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Val Ile Leu 860 865 870 875 ctc atg tgt gct gta cac ccg act ctg gta ttt gac atc acc aaa ttg 2992 Leu Met Cys Ala Val His Pro Thr Leu Val Phe Asp Ile Thr Lys Leu 880 885 890 ctg ctg gcc gtc ttc gga ccc ctt tgg att ctt caa gcc agt ttg ctt 3040 Leu Leu Ala Val Phe Gly Pro Leu Trp Ile Leu Gln Ala Ser Leu Leu 895 900 905 aaa gta ccc tac ttt gtg cgc gtc caa ggc ctt ctc cgg ttc tgc gcg 3088 Lys Val Pro Tyr Phe Val Arg Val Gln Gly Leu Leu Arg Phe Cys Ala 910 915 920 tta gcg cgg aag atg atc gga ggc cat tac gtg caa atg gtc atc att 3136 Leu Ala Arg Lys Met Ile Gly Gly His Tyr Val Gln Met Val Ile Ile 925 930 935 aag tta ggg gcg ctt act ggc acc tat gtt tat aac cat ctc act cct 3184 Lys Leu Gly Ala Leu Thr Gly Thr Tyr Val Tyr Asn His Leu Thr Pro 940 945 950 955 ctt cgg gac tgg gcg cac aac ggc ttg cga gat ctg gcc gtg gct gta 3232 Leu Arg Asp Trp Ala His Asn Gly Leu Arg Asp Leu Ala Val Ala Val 960 965 970 gag cca gtc gtc ttc tcc caa atg gag acc aag ctc atc acg tgg ggg 3280 Glu Pro Val Val Phe Ser Gln Met Glu Thr Lys Leu Ile Thr Trp Gly 975 980 985 gca gat acc gcc gcg tgc ggt gac atc atc aac ggc ttg cct gtt tcc 3328 Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Asn Gly Leu Pro Val Ser 990 995 1000 gcc cgc agg ggc cgg gag ata ctg ctc ggg cca gcc gat gga atg gtc 3376 Ala Arg Arg Gly Arg Glu Ile Leu Leu Gly Pro Ala Asp Gly Met Val 1005 1010 1015 tcc aag ggg tgg agg ttg ctg gcg ccc atc acg gcg tac gcc cag cag 3424 Ser Lys Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln 1020 1025 1030 1035 aca agg ggc ctc cta ggg tgc ata atc acc agc cta act ggc cgg gac 3472 Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp 1040 1045 1050 aaa aac caa gtg gag ggt gag gtc cag att gtg tca act gct gcc caa 3520 Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln 1055 1060 1065 acc ttc ctg gca acg tgc atc aat ggg gtg tgc tgg act gtc tac cac 3568 Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His 1070 1075 1080 ggg gcc gga acg agg acc atc gcg tca ccc aag ggt cct gtc atc cag 3616 Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln 1085 1090 1095 atg tat acc aat gta gac caa gac ctt gtg ggc tgg ccc gct ccg caa 3664 Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln 1100 1105 1110 1115 ggt agc cgc tca ttg aca ccc tgc act tgc ggc tcc tcg gac ctt tac 3712 Gly Ser Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr 1120 1125 1130 ctg gtc acg agg cac gcc gat gtc att ccc gtg cgc cgg cgg ggt gat 3760 Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp 1135 1140 1145 agc agg ggc agc ctg ctg tcg ccc cgg ccc att tcc tac ttg aaa ggc 3808 Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly 1150 1155 1160 tcc tcg ggg ggt ccg ctg ttg tgc ccc gcg ggg cac gcc gtg ggc ata 3856 Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile 1165 1170 1175 ttt agg gcc gcg gtg tgc acc cgt gga gtg gct aag gcg gtg gac ttt 3904 Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe 1180 1185 1190 1195 atc cct gtg gag aac cta gag aca acc atg agg tcc ccg gtg ttc acg 3952 Ile Pro Val Glu Asn Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr 1200 1205 1210 gat aac tcc tct cca cca gta gtg ccc cag agc ttc cag gtg gct cac 4000 Asp Asn Ser Ser Pro Pro Val Val Pro Gln Ser Phe Gln Val Ala His 1215 1220 1225 ctc cat gct ccc aca ggc agc ggc aaa agc acc aag gtc ccg gct gca 4048 Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala 1230 1235 1240 tat gca gct cag ggc tat aag gtg cta gta ctc aac ccc tct gtt gct 4096 Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala 1245 1250 1255 gca aca ctg ggc ttt ggt gct tac atg tcc aag gct cat ggg atc gat 4144 Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp 1260 1265 1270 1275 cct aac atc agg acc ggg gtg aga aca att acc act ggc agc ccc atc 4192 Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile 1280 1285 1290 acg tac tcc acc tac ggc aag ttc ctt gcc gac ggc ggg tgc tcg ggg 4240 Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly 1295 1300 1305 ggc gct tat gac ata ata att tgt gac gag tgc cac tcc acg gat gcc 4288 Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala 1310 1315 1320 aca tcc atc ttg ggc atc ggc act gtc ctt gac caa gca gag act gcg 4336 Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala 1325 1330 1335 ggg gcg aga ctg gtt gtg ctc gcc acc gcc acc cct ccg ggc tcc gtc 4384 Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val 1340 1345 1350 1355 act gtg ccc cat ccc aac atc gag gag gtt gct ctg tcc acc acc gga 4432 Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly 1360 1365 1370 gag atc cct ttt tac ggc aag gct atc ccc ctc gaa gta atc aag ggg 4480 Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val Ile Lys Gly 1375 1380 1385 ggg aga cat ctc atc ttc tgt cat tca aag aag aag tgc gac gaa ctc 4528 Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu 1390 1395 1400 gcc gca aag ctg gtc gca ttg ggc atc aat gcc gtg gcc tac tac cgc 4576 Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg 1405 1410 1415 ggt ctt gac gtg tcc gtc atc ccg acc agc ggc gat gtt gtc gtc gtg 4624 Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val 1420 1425 1430 1435 gca acc gat gcc ctc atg acc ggc tat acc ggc gac ttc gac tcg gtg 4672 Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val 1440 1445 1450 ata gac tgc aat acg tgt gtc acc cag aca gtc gat ttc agc ctt gac 4720 Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp 1455 1460 1465 cct acc ttc acc att gag aca atc acg ctc ccc cag gat gct gtc tcc 4768 Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro Gln Asp Ala Val Ser 1470 1475 1480 cgc act caa cgt cgg ggc agg act ggc agg ggg aag cca ggc atc tac 4816 Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr 1485 1490 1495 aga ttt gtg gca ccg ggg gag cgc ccc tcc ggc atg ttc gac tcg tcc 4864 Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser 1500 1505 1510 1515 gtc ctc tgt gag tgc tat gac gca ggc tgt gct tgg tat gag ctc acg 4912 Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr 1520 1525 1530 ccc gcc gag act aca gtt agg cta cga gcg tac atg aac acc ccg ggg 4960 Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly 1535 1540 1545 ctt ccc gtg tgc cag gac cat ctt gaa ttt tgg gag ggc gtc ttt aca 5008 Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr 1550 1555 1560 ggc ctc act cat ata gat gcc cac ttt cta tcc cag aca aag cag agt 5056 Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser 1565 1570 1575 ggg gag aac ctt cct tac ctg gta gcg tac caa gcc acc gtg tgc gct 5104 Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala 1580 1585 1590 1595 agg gct caa gcc cct ccc cca tcg tgg gac cag atg tgg aag tgt ttg 5152 Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu 1600 1605 1610 att cgc ctc aag ccc acc ctc cat ggg cca aca ccc ctg cta tac aga 5200 Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg 1615 1620 1625 ctg ggc gct gtt cag aat gaa atc acc ctg acg cac cca gtc acc aaa 5248 Leu Gly Ala Val Gln Asn Glu Ile Thr Leu Thr His Pro Val Thr Lys 1630 1635 1640 tac atc atg aca tgc atg tcg gcc gac ctg gag gtc gtc acg agc acc 5296 Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr 1645 1650 1655 tgg gtg ctc gtt ggc ggc gtc ctg gct gct ttg gcc gcg tat tgc ctg 5344 Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu 1660 1665 1670 1675 tca aca ggc tgc gtg gtc ata gtg ggc agg gtc gtc ttg tcc ggg aag 5392 Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu Ser Gly Lys 1680 1685 1690 ccg gca atc ata cct gac agg gaa gtc ctc tac cga gag ttc gat gag 5440 Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu 1695 1700 1705 atg gaa gag tgc tct cag cac tta ccg tac atc gag caa ggg atg atg 5488 Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met 1710 1715 1720 ctc gcc gag cag ttc aag cag aag gcc ctc ggc ctc ctg cag acc gcg 5536 Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala 1725 1730 1735 tcc cgt cag gca gag gtt atc gcc cct gct gtc cag acc aac tgg caa 5584 Ser Arg Gln Ala Glu Val Ile Ala Pro Ala Val Gln Thr Asn Trp Gln 1740 1745 1750 1755 aaa ctc gag acc ttc tgg gcg aag cat atg tgg aac ttc atc agt ggg 5632 Lys Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly 1760 1765 1770 ata caa tac ttg gcg ggc ttg tca acg ctg cct ggt aac ccc gcc att 5680 Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile 1775 1780 1785 gct tca ttg atg gct ttt aca gct gct gtc acc agc cca cta acc act 5728 Ala Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr 1790 1795 1800 agc caa acc ctc ctc ttc aac ata ttg ggg ggg tgg gtg gct gcc cag 5776 Ser Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln 1805 1810 1815 ctc gcc gcc ccc ggt gcc gct act gcc ttt gtg ggc gct ggc tta gct 5824 Leu Ala Ala Pro Gly Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala 1820 1825 1830 1835 ggc gcc gcc atc ggc agt gtt gga ctg ggg aag gtc ctc ata gac atc 5872 Gly Ala Ala Ile Gly Ser Val Gly Leu Gly Lys Val Leu Ile Asp Ile 1840 1845 1850 ctt gca ggg tat ggc gcg ggc gtg gcg gga gct ctt gtg gca ttc aag 5920 Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys 1855 1860 1865 atc atg agc ggt gag gtc ccc tcc acg gag gac ctg gtc aat cta ctg 5968 Ile Met Ser Gly Glu Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu 1870 1875 1880 ccc gcc atc ctc tcg ccc gga gcc ctc gta gtc ggc gtg gtc tgt gca 6016 Pro Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala 1885 1890 1895 gca ata ctg cgc cgg cac gtt ggc ccg ggc gag ggg gca gtg cag tgg 6064 Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp 1900 1905 1910 1915 atg aac cgg ctg ata gcc ttc gcc tcc cgg ggg aac cat gtt tcc ccc 6112 Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro 1920 1925 1930 acg cac tac gtg ccg gag agc gat gca gct gcc cgc gtc act gcc ata 6160 Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Ala Ile 1935 1940 1945 ctc agc agc ctc act gta acc cag ctc ctg agg cga ctg cac cag tgg 6208 Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp 1950 1955 1960 ata agc tcg gag tgt acc act cca tgc tcc ggt tcc tgg cta agg gac 6256 Ile Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp 1965 1970 1975 atc tgg gac tgg ata tgc gag gtg ttg agc gac ttt aag acc tgg cta 6304 Ile Trp Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu 1980 1985 1990 1995 aaa gct aag ctc atg cca cag ctg cct ggg atc ccc ttt gtg tcc tgc 6352 Lys Ala Lys Leu Met Pro Gln Leu Pro Gly Ile Pro Phe Val Ser Cys 2000 2005 2010 cag cgc ggg tat aag ggg gtc tgg cga gtg gac ggc atc atg cac act 6400 Gln Arg Gly Tyr Lys Gly Val Trp Arg Val Asp Gly Ile Met His Thr 2015 2020 2025 cgc tgc cac tgt gga gct gag atc act gga cat gtc aaa aac ggg acg 6448 Arg Cys His Cys Gly Ala Glu Ile Thr Gly His Val Lys Asn Gly Thr 2030 2035 2040 atg agg atc gtc ggt cct agg acc tgc agg aac atg tgg agt ggg acc 6496 Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp Ser Gly Thr 2045 2050 2055 ttc ccc att aat gcc tac acc acg ggc ccc tgt acc ccc ctt cct gcg 6544 Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Leu Pro Ala 2060 2065 2070 2075 ccg aac tac acg ttc gcg cta tgg agg gtg tct gca gag gaa tat gtg 6592 Pro Asn Tyr Thr Phe Ala Leu Trp Arg Val Ser Ala Glu Glu Tyr Val 2080 2085 2090 gag ata agg cag gtg ggg gac ttc cac tac gtg acg ggt atg act act 6640 Glu Ile Arg Gln Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr 2095 2100 2105 gac aat ctc aaa tgc ccg tgc cag gtc cca tcg ccc gaa ttt ttc aca 6688 Asp Asn Leu Lys Cys Pro Cys Gln Val Pro Ser Pro Glu Phe Phe Thr 2110 2115 2120 gaa ttg gac ggg gtg cgc cta cat agg ttt gcg ccc ccc tgc aag ccc 6736 Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pro 2125 2130 2135 ttg ctg cgg gag gag gta tca ttc aga gta gga ctc cac gaa tac ccg 6784 Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu His Glu Tyr Pro 2140 2145 2150 2155 gta ggg tcg caa tta cct tgc gag ccc gaa ccg gac gtg gcc gtg ttg 6832 Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val Ala Val Leu 2160 2165 2170 acg tcc atg ctc act gat ccc tcc cat ata aca gca gag gcg gcc ggg 6880 Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Gly 2175 2180 2185 cga agg ttg gcg agg gga tca ccc ccc tct gtg gcc agc tcc tcg gct 6928 Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val Ala Ser Ser Ser Ala 2190 2195 2200 agc cag cta tcc gct cca tct ctc aag gca act tgc acc gct aac cat 6976 Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn His 2205 2210 2215 gac tcc cct gat gct gag ctc ata gag gcc aac ctc cta tgg agg cag 7024 Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln 2220 2225 2230 2235 gag atg ggc ggc aac atc acc agg gtt gag tca gaa aac aaa gtg gtg 7072 Glu Met Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Val Val 2240 2245 2250 att ctg gac tcc ttc gat ccg ctt gtg gcg gag gag gac gag cgg gag 7120 Ile Leu Asp Ser Phe Asp Pro Leu Val Ala Glu Glu Asp Glu Arg Glu 2255 2260 2265 atc tcc gta ccc gca gaa atc ctg cgg aag tct cgg aga ttc gcc cag 7168 Ile Ser Val Pro Ala Glu Ile Leu Arg Lys Ser Arg Arg Phe Ala Gln 2270 2275 2280 gcc ctg ccc gtt tgg gcg cgg ccg gac tat aac ccc ccg cta gtg gag 7216 Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu 2285 2290 2295 acg tgg aaa aag ccc gac tac gaa cca cct gtg gtc cat ggc tgt ccg 7264 Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His Gly Cys Pro 2300 2305 2310 2315 ctt cca cct cca aag tcc cct cct gtg cct ccg cct cgg aag aag cgg 7312 Leu Pro Pro Pro Lys Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Arg 2320 2325 2330 acg gtg gtc ctc act gaa tca acc cta tct act gcc ttg gcc gag ctc 7360 Thr Val Val Leu Thr Glu Ser Thr Leu Ser Thr Ala Leu Ala Glu Leu 2335 2340 2345 gcc acc aga agc ttt ggc agc tcc tca act tcc ggc att acg ggc gac 7408 Ala Thr Arg Ser Phe Gly Ser Ser Ser Thr Ser Gly Ile Thr Gly Asp 2350 2355 2360 aat acg aca aca tcc tct gag ccc gcc cct tct ggc tgc ccc ccc gac 7456 Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser Gly Cys Pro Pro Asp 2365 2370 2375 tcc gac gct gag tcc tat tcc tcc atg ccc ccc ctg gag ggg gag cct 7504 Ser Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro 2380 2385 2390 2395 ggg gat ccg gat ctt agc gac ggg tca tgg tca acg gtc agt agt gag 7552 Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr Val Ser Ser Glu 2400 2405 2410 gcc aac gcg gag gat gtc gtg tgc tgc tca atg tct tac tct tgg aca 7600 Ala Asn Ala Glu Asp Val Val Cys Cys Ser Met Ser Tyr Ser Trp Thr 2415 2420 2425 ggc gca ctc gtc acc ccg tgc gcc gcg gaa gaa cag aaa ctg ccc atc 7648 Gly Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gln Lys Leu Pro Ile 2430 2435 2440 aat gca cta agc aac tcg ttg cta cgt cac cac aat ttg gtg tat tcc 7696 Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Leu Val Tyr Ser 2445 2450 2455 acc acc tca cgc agt gct tgc caa agg cag aag aaa gtc aca ttt gac 7744 Thr Thr Ser Arg Ser Ala Cys Gln Arg Gln Lys Lys Val Thr Phe Asp 2460 2465 2470 2475 aga ctg caa gtt ctg gac agc cat tac cag gac gta ctc aag gag gtt 7792 Arg Leu Gln Val Leu Asp Ser His Tyr Gln Asp Val Leu Lys Glu Val 2480 2485 2490 aaa gca gcg gcg tca aaa gtg aag gct aac ttg cta tcc gta gag gaa 7840 Lys Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser Val Glu Glu 2495 2500 2505 gct tgc agc ctg acg ccc cca cac tca gcc aaa tcc aag ttt ggt tat 7888 Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr 2510 2515 2520 ggg gca aaa gac gtc cgt tgc cat gcc aga aag gcc gta acc cac atc 7936 Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Thr His Ile 2525 2530 2535 aac tcc gtg tgg aaa gac ctt ctg gaa gac aat gta aca cca ata gac 7984 Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val Thr Pro Ile Asp 2540 2545 2550 2555 act acc atc atg gct aag aac gag gtt ttc tgc gtt cag cct gag aag 8032 Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys 2560 2565 2570 ggg ggt cgt aag cca gct cgt ctc atc gtg ttc ccc gat ctg ggc gtg 8080 Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val 2575 2580 2585 cgc gtg tgc gaa aag atg gct ttg tac gac gtg gtt aca aag ctc ccc 8128 Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr Lys Leu Pro 2590 2595 2600 ttg gcc gtg atg gga agc tcc tac gga ttc caa tac tca cca gga cag 8176 Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gln Tyr Ser Pro Gly Gln 2605 2610 2615 cgg gtt gaa ttc ctc gtg caa gcg tgg aag tcc aag aaa acc cca atg 8224 Arg Val Glu Phe Leu Val Gln Ala Trp Lys Ser Lys Lys Thr Pro Met 2620 2625 2630 2635 ggg ttc tcg tat gat acc cgc tgc ttt gac tcc aca gtc act gag agc 8272 Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Ser 2640 2645 2650 gac atc cgt acg gag gag gca atc tac caa tgt tgt gac ctc gac ccc 8320 Asp Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp Leu Asp Pro 2655 2660 2665 caa gcc cgc gtg gcc atc aag tcc ctc acc gag agg ctt tat gtt ggg 8368 Gln Ala Arg Val Ala Ile Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly 2670 2675 2680 ggc cct ctt acc aat tca agg ggg gag aac tgc ggc tat cgc agg tgc 8416 Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys 2685 2690 2695 cgc gcg agc ggc gta ctg aca act agc tgt ggt aac acc ctc act tgc 8464 Arg Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys 2700 2705 2710 2715 tac atc aag gcc cgg gca gcc tgt cga gcc gca ggg ctc cag gac tgc 8512 Tyr Ile Lys Ala Arg Ala Ala Cys Arg Ala Ala Gly Leu Gln Asp Cys 2720 2725 2730 acc atg ctc gtg tgt ggc gac gac tta gtc gtt atc tgt gaa agc gcg 8560 Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala 2735 2740 2745 ggg gtc cag gag gac gcg gcg agc ctg aga gcc ttc acg gag gct atg 8608 Gly Val Gln Glu Asp Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Met 2750 2755 2760 acc agg tac tcc gcc ccc cct ggg gac ccc cca caa cca gaa tac gac 8656 Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp 2765 2770 2775 ttg gag ctc ata aca tca tgc tcc tcc aac gtg tca gtc gcc cac gac 8704 Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His Asp 2780 2785 2790 2795 ggc gct gga aag agg gtc tac tac ctc acc cgt gac cct aca acc ccc 8752 Gly Ala Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro 2800 2805 2810 ctc gcg aga gct gcg tgg gag aca gca aga cac act cca gtc aat tcc 8800 Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn Ser 2815 2820 2825 tgg cta ggc aac ata atc atg ttt gcc ccc aca ctg tgg gcg agg atg 8848 Trp Leu Gly Asn Ile Ile Met Phe Ala Pro Thr Leu Trp Ala Arg Met 2830 2835 2840 ata ctg atg acc cat ttc ttt agc gtc ctt ata gcc agg gac cag ctt 8896 Ile Leu Met Thr His Phe Phe Ser Val Leu Ile Ala Arg Asp Gln Leu 2845 2850 2855 gaa cag gcc ctc gat tgc gag atc tac ggg gcc tgc tac tcc ata gaa 8944 Glu Gln Ala Leu Asp Cys Glu Ile Tyr Gly Ala Cys Tyr Ser Ile Glu 2860 2865 2870 2875 cca ctt gat cta cct cca atc att caa aga ctc cat ggc ctc agc gca 8992 Pro Leu Asp Leu Pro Pro Ile Ile Gln Arg Leu His Gly Leu Ser Ala 2880 2885 2890 ttt tca ctc cac agt tac tct cca ggt gaa att aat agg gtg gcc gca 9040 Phe Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Ala 2895 2900 2905 tgc ctc aga aaa ctt ggg gta ccg ccc ttg cga gct tgg aga cac cgg 9088 Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp Arg His Arg 2910 2915 2920 gcc cgg agc gtc cgc gct agg ctt ctg gcc aga gga ggc agg gct gcc 9136 Ala Arg Ser Val Arg Ala Arg Leu Leu Ala Arg Gly Gly Arg Ala Ala 2925 2930 2935 ata tgt ggc aag tac ctc ttc aac tgg gca gta aga aca aag ctc aaa 9184 Ile Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys 2940 2945 2950 2955 ctc act cca ata gcg gcc gct ggc cag ctg gac ttg tcc ggc tgg ttc 9232 Leu Thr Pro Ile Ala Ala Ala Gly Gln Leu Asp Leu Ser Gly Trp Phe 2960 2965 2970 acg gct ggc tac agc ggg gga gac att tat cac agc gtg tct cat gcc 9280 Thr Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Val Ser His Ala 2975 2980 2985 cgg ccc cgc tgg atc tgg ttt tgc cta ctc ctg ctt gct gca ggg gta 9328 Arg Pro Arg Trp Ile Trp Phe Cys Leu Leu Leu Leu Ala Ala Gly Val 2990 2995 3000 ggc atc tac ctc ctc ccc aac cga tgaaggttgg ggtaaacact ccggcct 9379 Gly Ile Tyr Leu Leu Pro Asn Arg 3005 3010 2 3011 PRT Virus 2 Met Ser Thr Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 

What is claimed is:
 1. A fusion polypeptide that comprises: (a) a first region consisting essentially of the C polypeptide of the hepatitis C virus (HCV) or a portion thereof that comprises a polypeptide region responsible for gene regulatory activity; and (b) a second region consisting essentially of the envelope polypeptide (E1) of the virus or a portion thereof that comprises a site for cytoplasmic anchorage of the E1 polypeptide; wherein the first region is fused by a peptide bond to the second region, and the fusion polypeptide is not cleaved by a mammalian protease.
 2. The polypeptide of claim 1, wherein the first region comprises a C-terminal end, and the second region comprises a N-terminal end, and the C-terminal end of the first region is fused with peptide bond to the N-terminal end of the second region.
 3. The fusion polypeptide of claim 2, wherein the fusion polypeptide does not contain the amino acid sequence Serine₁₉₀-Alanine₁₉₁-Tyrosine₁₉₂-Glutamic acid_(193.)
 4. The fusion polypeptide of claim 3, that contains an amino acid sequence that corresponds to the amino acid sequence Serine₁₉₀-Alanine₁₉₁-Tyrosine₁₉₂-Glutamic acid₁₉₃ wherein the Alanine residue is substituted by a Valine residue or wherein the Tyrosine residue is substituted by an Asparagine residue.
 5. The fusion polypeptide of claim 1, wherein the amino acid sequence of the first region does not contain the amino acid sequence Cysteine₁₇₂-Serine₁₇₃-Phenylalanine₁₇₄-Serine_(175.)
 6. The fusion polypeptide of claim 1, wherein the second region further comprises the amino acid sequence of polypeptide E1 from amino acid No. 194 to amino acid No.
 380. 7. The fusion polypeptide of claim 1, wherein the second region further comprises an amino acid sequence selected in the group consisting of amino acid Nos 262 to 291, amino acid Nos 370 to 380, amino acid Nos 330 to 380 of the E1 envelope polypeptide.
 8. The fusion polypeptide of claim 1, wherein the first region comprises the amino acid sequence of the C polypeptide from amino acid in position 1 to any amino acid in position 48 to
 191. 9. The fusion polypeptide of claim 8, wherein the first region comprises the amino acid sequence of the C polypeptide from amino acid in position 1 to amino acid in position
 120. 10. A fusion polypeptide comprising: a) a C polypeptide of HCV or a portion thereof that comprises: 1) a first C polypeptide region responsible for gene regulatory activity and 2) a second C polypeptide region responsible for the interaction with the E1 envelope polypeptide, wherein the site of interaction with the E1 polypeptide is between about 151 and about 173, or between about 173 and about 191, and b) a E1 envelope polypeptide of HCV or a portion thereof that comprises: 1) a first E1 polypeptide region responsible for E1 cytoplasmic anchorage and 2) a second E1 polypeptide region responsible for the interaction of said second C polypeptide region, wherein the site for interaction with the C polypeptide is between about amino acid 330 and about amino acid 380; wherein said C polypeptide is fused by a peptide bond to said E1 envelope polypeptide, and the C polypeptide comprises Cysteine₁₇₂-Serine₁₇₃-Phenylalanine₁₇₄-Serine₁₇₅ with at least one mutation between amino acid Nos 172 and
 175. 11. The fusion polypeptide of claim 10, wherein the Serine residue in position 173 is substituted by a Methionine residue or wherein the Phenylalanine residue is substituted by a Leucine residue.
 12. The fusion polypeptide of claim 10, wherein the C polypeptide contains a C terminal end and the E1 polypeptide contains a N terminal end, wherein the C terminal end is fused to the N terminal end and, wherein the fusion polypeptide contains the amino acid sequence Serine₁₉₀-Alanine₁₉₁-Tyrosine₁₉₂-Glutamic acid_(193.)
 13. The fusion polypeptide of claim 10, wherein the E1 envelope polypeptide comprises the amino acid sequence of polypeptide E1 from amino acid in position 192 to amino acid in position
 380. 14. The fusion polypeptide of claim 10, wherein the C polypeptide comprises the amino acid sequence of the C polypeptide from amino acid in position 1 to amino acid in position
 171. 15. The fusion polypeptide of claim 14, wherein the C polypeptide further comprises the amino acid sequence of the C polypeptide from amino acid in position 176 to amino acid in position
 191. 